Small-molecule metabolic and signaling pathways regulate nearly all cellular and physiological activities. Alterations in these biochemical networks likely contribute to the development of several diseases, including cancer. Nonetheless, the discovery and functional characterization of biochemical pathways that support cancer have, to date, been hindered by a lack of technologies that can globally inventory their molecular components in biological systems. To address this central problem, we have introduced a set of chemical proteomic and metabolomic technologies termed activity-based protein profiling (ABPP) and metabolite enrichment by tagging and proteolytic release (METPR), respectively. ABPP, which utilizes active site-directed chemical probes to record the functional state of enzymes directly in native proteomes, has led to the discovery of enzyme activities that are elevated in aggressive cancer cells. Several of these enzymes are completely unannotated, suggesting that they regulate novel metabolic pathways in cancer. We have shown that ABPP, when coupled with untargeted metabolite profiling, facilitates assignment of biochemical functions to unannotated enzymes that support cancer pathogenesis. METPR is an advanced metabolomics method that employs chemical probes to tag, enrich, and profile endogenous small molecules of any physicochemical class. In this application, we propose to unite our enzyme and small molecule profiling methods to create the first integrated "systems biology" platform for the global analysis of biochemical pathways altered in human cancer cells. Specifically, we will: 1) identify enzyme activities and metabolites that are elevated in aggressive cancer cells, 2) assemble these biomolecules into metabolic pathways and test their function in cancer, and 3) confirm that these pathogenesis-related biochemical pathways are dysregulated in primary human cancers. Collectively, these studies should yield fundamental insights into the molecular basis of cancer and, at the same time, achieve several of the overarching goals of this RFA, including determining: 1) how pathways are differentially regulated in disease states (i.e., aggressive versus non-aggressive cancer cells), 2) new connections between pathways (i.e., annotation of uncharacterized enzymes that regulate biochemical networks in cancer), and 3) the suitability of innovative metabolomic technologies (i.e., METPR) for basic and translational applications.